Biotin, or vitamin H, is an indispensable element in intermediary metabolism in many organisms since it is an essential factor of biotin-dependent carboxylases important in fatty acid synthesis, gluconeogenesis, and amino acid metabolism. Biotin is useful as a food supplement, as a cosmetic additive, and as a diagnostic reagent in biotin-avidin-based detection assays.
Most biotin for commercial use is currently produced by a complex chemical synthesis process. Although several investigators are attempting to synthesize biotin in commercial quantities using microbiological methods, the cost thus far has been prohibitive. Wild type microorganisms produce only small amounts of the vitamin apparently because such microorganisms exert tight control over biotin biosynthesis. In an effort to improve microbial biotin production, some investigators have transformed microorganisms with Escherichia coli or Bacillus sphaericus genes that encode certain proteins involved in the biotin biosynthetic pathway. Although expression of these genes in some cases did increase true biotin and/or biotin vitamer production, the amount of true biotin produced using such methods is substantially lower than that required for a commercially viable process.
The biotin biosynthetic pathway in Escherichia coli is thought to include at least 5 enzymatic steps catalyzed by enzymes encoded by Escherichia coli bioA, bioB, bioF, bioC, and bioD genes contained on the biotin operon. The Escherichia coli bioA, bioB, bioD, and bioF genes are thought to encode enzymes having the following respective activities: 7,8-diaminopelargonic acid aminotransferase (also called 7,8-diaminopelargonic acid synthase), biotin synthetase (also called biotin synthase), desthiobiotin synthetase (also called desthiobiotin synthase), and 7-keto-8-aminopelargonic acid synthetase (also called 7-keto-8-aminopelargonic acid synthase). The protein encoded by the Escherichia coli bioC gene is thought to operate at an early step in the biotin biosynthetic pathway, but the protein's actual function is presently unknown. The biotin operon also includes an additional open reading frame, referred to as Escherichia coli ORF 1, the function of which, until the present invention, has been unknown (e.g., Otsuka et al., pp. 19577-19585, 1988, J. Biol. Chem., vol. 263; Brown et al., pp. 295-326, 1991, Biotech. Genet. Engineer. Reviews, vol. 9; Eisenberg, pp. 544-550, 1987, in Escherichia coli and Salmonella typhimurium Cellular and Molecular Biology, Neidhardt, F. C. et al., eds., American Society of Microbiology, Washington, D.C.). In addition, the Escherichia coli bioH gene, located at a site distant from the biotin operon, encodes a protein thought to be involved in an early, but as yet unknown, step in the biotin biosynthetic pathway (e.g., O'Regan et al., p. 8004, 1989, Nucleic Acids Res., vol 17; Brown et al., ibid.
Two gene clusters encoding enzymes involved in biotin biosynthesis have been isolated from Bacillus sphaericus. The two gene clusters include the linked Bacillus sphaericus genes bioD, bioA, bioY, and bioB, also referred to as Bacillus sphaericus bioDAYB; and linked Bacillus sphaericus genes bioX, bioW, and bioF, also referred to as Bacillus sphaericus bioXWF (see, for example, Gloeckler et al., pp. 63-70, 1990, Gene, vol. 87; U.S. Pat. No. 5,096,823 by Gloeckler et al., issued Mar. 17, 1992; European Patent Office Publication No. 266,240, by Gloeckler et al., published May 4, 1988; and European Patent Publication No. 240,105, by Ohsawa et al., published Nov. 7, 1987). Bacillus sphaericus and Escherichia coli bioA, bioB, bioD, and bioF genes are structurally similar and apparently encode functionally equivalent enzymes (e.g., Brown et al., ibid.). Bacillus sphaericus bioW, bioX and bioY genes, which apparently are not structurally homologous to known Escherichia coli genes, are thought to be involved in the active uptake of pimelic acid by Bacillus sphaericus (e.g., Brown et al., ibid.). In contrast, some investigators have hypothesized that uptake of pimelic acid by Escherichia coli is by passive diffusion (e.g., Brown et al., ibid.; Ploux et al., pp. 685-690, 1992, Biochem. J., vol. 287).
Several investigators have disclosed systems to attempt to express biotin using the Escherichia coli biotin operon. For example, GB Publication No. 2,216,530, by Pearson et al., published Oct. 11, 1989, discloses expression of the Escherichia coli biotin operon in Saccharomyces cerevisiae but does not report biotin production levels. In another example, Fisher, in U.S. Pat. No. 5,110,731, issued May 5, 1992, discloses that a biotin retention-deficient mutant of Escherichia coli transformed with a plasmid containing the Escherichia coli biotin operon produced a maximum of 30 milligrams (mg) of biotin per liter of medium.
Several researchers (see, for example, Ogata, pp. 390-394, 1970, Methods in Enzymology, vol. 17a; Izumi et al., pp. 231-256, in Biotechnology of Vitamins, Pigments, and Growth Factors, Elsevier Applied Science, E. J. Vandamme, ed.; U.S. Pat. No. 3,393,129, by Shibata et al., issued Jul. 16, 1968; and U.S. Pat. No. 4,563,426 by Yamada et al., issued Jan. 7, 1986) have reported that true biotin and biotin vitamer production by fungal and bacterial microorganisms, and in particular by Bacillus sphaericus, increases when the microorganisms are grown in the presence of biotin precursors, such as pimelic acid and desthiobiotin. Based upon this observation, attempts have been made to increase biotin production by transforming Escherichia coli and Bacillus sphaericus microorganisms with either the Bacillus sphaericus bioB gene or Bacillus sphaericus bioDAYB and bioXWF biotin gene clusters and growing the transformants in the presence of biotin precursors.
European Patent Publication No. 375,525, by Gloeckler et al., published Jun. 27, 1990, discloses the use of Escherichia coli host cells transformed with the two clusters of Bacillus sphaericus biotin operon genes (i.e., bioDAYB and bioXWF) to produce biotin. When such transformed hosts were grown in medium containing pimelic acid, they produced 144-160 mg of biotin vitamers per liter of medium but only 15-16 mg of true biotin per liter of medium. Thus, the amount of true biotin produced was only about 9 to 10 percent of the amount of total biotin (i.e., true biotin and vitamers) produced, indicating that, despite a high gene copy number, the transformed cells could not completely convert the biotin vitamers to true biotin. In addition, of the total amount of biotin vitamers produced, only 25 percent to 28 percent was desthiobiotin (the direct precursor of biotin), suggesting that about 70 percent of the biotin vitamers produced were compounds that had yet to be converted to desthiobiotin.
Sabatie et al., pp.29-50, 1991, Journal of Biotechnology, vol. 20, also transformed Escherichia coli cells with a vector containing the Bacillus sphaericus bioDAYB and bioXWF gene clusters. When such transformed cells were grown in the presence of pimelic acid under fed-batch fermentation conditions, the cells produced 300 mg of biotin vitamers per liter of medium, but only 45 mg of true biotin per liter of medium. Thus, the amount of true biotin produced by Sabatie et al. was only 13 percent of the total amount of biotin (i.e., true biotin and vitamers) produced, again indicating inefficient conversion of biotin vitamers to true biotin.
Ohsawa et al., pp. 39-48, 1989, Gene, vol. 80, transformed Escherichia coli, Bacillus sphaericus and Bacillus subtilis with vectors containing the Bacillus sphaericus bioB gene under the control of suitable promoters. Transformed strains were grown in medium containing desthiobiotin. Biotin production by Escherichia coli and Bacillus subtilis cells transformed with plasmids containing the Bacillus sphaericus bioB gene was about 1500-fold higher than biotin production by cells transformed with plasmids lacking the Bacillus sphaericus bioB gene. Biotin production by Bacillus sphaericus cells transformed with plasmids containing the Bacillus sphaericus bioB gene was about 100-fold higher than biotin production by cells transformed with plasmids lacking the Bacillus sphaericus bioB gene.
Ohsawa et al., pp. 121-124, 1992, J. Ferment. Bioeng., vol. 73, also cultured Bacillus sphaericus cells transformed with a plasmid containing the Bacillus sphaericus bioB gene in medium containing pimelic acid. Cells transformed with a plasmid lacking the Bacillus sphaericus bioB gene made less than 0.2 mg of true biotin per liter of medium and about 25 mg of vitamers and true biotin per liter of medium. Cells transformed with a plasmid containing the Bacillus sphaericus bioB gene made about 1.2-3.5 mg of true biotin per liter of medium and about 30 mg of vitamers and true biotin per liter of medium. Thus, despite the increased expression of the Bacillus sphaericus bioB gene, only 4 percent to 10.4 percent of the total amount of biotin (i.e., biotin vitamers and true biotin) produced was true biotin.
Additional attempts to increase biotin production have included efforts to obtain hosts that are derepressed for biotin synthesis (see, for example, Japanese Patent Publication No. 62,155,081, assigned to Shiseido KK, published Jul. 10, 1987; Japanese Patent Publication No. 61,202,686, assigned to Shiseido KK, published Sep. 8, 1986; Japanese Patent Publication No. 61,149,091, assigned to Nippon Soda KK, published Jul. 7, 1986; and European Patent Publication No. 379,442, by Gloeckler et al., published Jul. 25, 1990), and to obtain low-acetate synthesizing mutants (see, for example, European Patent Publication No. 316,229, by Haze et al., published May 17, 1989). However, none of these techniques has led to the production of commercially significant amounts of true biotin.
Thus there remains both a need to improve overall biotin production by amplifying expression of additional genes in the biotin biosynthetic pathway and to improve production of true biotin by engineering cells to convert biotin vitamers to true biotin.